RESUMO
The helminth Trichostrongylus colubriformis is a parasitic nematode infecting the small intestine of sheep. We report the isolation and characterization of a 30-kDa glycoprotein capable of partially protecting guinea-pigs against the parasite. This glycoprotein is secreted by the L4 and adult parasitic stages of the worm. The sequence of three separate cDNA clones predicts the polypeptide to be about 15 kDa, with four N-linked carbohydrate chains and an internal disulphide bond. The clones also indicate the existence of sequence variability in this antigen. Limited sequence homology to a porcine intestinal peptide suggests an influence on host gut physiology.
Assuntos
Antígenos de Helmintos/imunologia , Glicoproteínas/imunologia , Proteínas de Helminto/imunologia , Tricostrongiloidíase/imunologia , Tricostrongilose/imunologia , Trichostrongylus/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Helmintos/genética , Antígenos de Helmintos/isolamento & purificação , Sequência de Bases , Northern Blotting , Clonagem Molecular , DNA/genética , Dissulfetos , Escherichia coli/genética , Glicoproteínas/genética , Glicoproteínas/isolamento & purificação , Cobaias , Proteínas de Helminto/genética , Proteínas de Helminto/isolamento & purificação , Dados de Sequência Molecular , RNA Mensageiro/biossíntese , Trichostrongylus/genética , Trichostrongylus/metabolismo , Vacinas/imunologiaRESUMO
The transforming gene of the Abelson murine leukaemia virus, v-abl, contains two open reading frames (orf). The 5' orf encodes a tyrosine-specific protein kinase while the 3' orf has the capacity to code for an 18,000 Mr protein. However, no 3' orf product has yet been identified. Using probes capable of distinguishing between the 5' and 3' orfs of v-abl, we have examined the abl-related transcripts present in human haematopoietic cells and leukaemia-derived cell lines, including the chronic myeloid leukaemia-derived cell line K562. Our results indicate that transcripts of 6 kb, 7 kb and 8 kb (kilobase, 10(3) base-pairs) show strong homology to v-abl 5' protein kinase-encoding orf sequences, but are devoid of any sequences from the v-abl 3' orf. In addition, transcripts of 5 kb, 3 kb, 1.6 kb and 1.4 kb, reacting with both 5' orf and 3' orf probes, were observed. The latter species, with coding sequences from both the tyrosine kinase and the putative 18,000 Mr protein, must be transcribed from the human c-abl gene as this is apparently the only human gene containing sequences homologous to the v-abl 3' orf. The 6 kb, 7 kb and 8 kb transcripts may arise either from the c-abl gene through differential splicing, or from one of the three other regions of the human genome with sequences homologous to the 5' orf of v-abl. Examination of genomic DNA from the K562 cell line revealed that the amplification of abl-related sequences, which is presumed to result in the elevated levels of the 8 kb transcript found in this cell line, does not involve sequences homologous to the v-abl 3' orf. This lends credence to the idea that the 8 kb transcript may derive from an abl-related gene other than c-abl. While the significance of the 3' orf of v-abl remains unknown, the data presented strongly suggest the existence of at least two distinct abl-related proteins in human haematopoietic cells.
Assuntos
Vírus da Leucemia Murina de Abelson/genética , Genes Virais , Sistema Hematopoético/microbiologia , Vírus da Leucemia Murina/genética , Animais , Linhagem Celular , Amplificação de Genes , Humanos , Leucemia Mieloide/genética , Leucemia Mieloide/microbiologia , Camundongos , Hibridização de Ácido Nucleico , Proteínas Tirosina Quinases/genética , RNA/genética , Transcrição Gênica , Proteínas Virais/genéticaRESUMO
Chicken erythroblasts can be transformed by the avian retrovirus, avian erythroblastosis virus (AEV). Earlier studies have shown that the mechanism of transformation appears to involve a "block" in differentiation, in that when erythroblasts are transformed by a temperature-sensitive mutant of ts34 AEV and incubated at the nonpermissive temperature, the cells start to differentiate and produce hemoglobin. We have decided to use this system to isolate pure populations of chicken erythroblasts and raise monoclonal antibodies against their cell surface proteins. Three monoclonal antibodies were isolated and tested for their ability to bind to various hematopoietic cell types; two were shown to be erythroid-specific, whereas the other antibody bound to proliferating cells but not to erythrocytes or granulocytes. Of the erythroid-specific antibodies, one precipitated a 94,000 molecular weight protein, whereas the other precipitated a 11,000 molecular weight protein that was tentatively identified as hemoglobin. The use of this system and approach to identify and evaluate changes that occur during the differentiation is discussed.
Assuntos
Antígenos de Superfície/genética , Eritroblastos/imunologia , Eritrócitos/imunologia , Animais , Anticorpos Monoclonais , Vírus da Mieloblastose Aviária/imunologia , Medula Óssea/imunologia , Diferenciação Celular , Transformação Celular Viral , Células Cultivadas , Galinhas , Imunofluorescência , Hibridomas/imunologiaRESUMO
Chicken erythroblasts transformed by a temperature-sensitive mutant of avian erythroblastosis virus (ts34 AEV) have a greatly increased haemoglobin content (Graf, T., N. Ade and H. Beug: Nature 275, 496-501 (1978)) if allowed to grow for 3-5 days at the non-permissive temperature (41 degrees C), instead of the permissive temperature (35 degrees C) of the virus. Cell-surface molecular changes during this differentiation were investigated by examining the glycoproteins synthesized by a ts34-transformed erythroblast cell line. These cells synthesized a greatly increased amount of a 94,000 molecular weight erythrocyte cell-surface glycoprotein beginning 2-6 h after a shift in growth temperature from 35 degrees to 41 degrees C, consistent with the proposal that such a shift releases these transformed cells from a differentiation block.